Pathogenic bacteria must assemble and secrete virulence factors to interact with host tissues and cause disease. Gram-negative bacteria have an outer membrane in addition to a cytoplasmic membrane and must secrete virulence factors across both these barriers. The mechanisms by which this occurs can be quite complex and are not well understood. The chaperone/usher pathway is a virulence protein secretion pathway that requires two components for secretion across the outer membrane: a periplasmic chaperone and an outer membrane protein termed an usher. The chaperone directs proper folding of the secreted proteins and prevents off-pathway interactions. The usher serves as an assembly platform at the outer membrane and provides a secretion channel to the cell surface. The chaperone/usher pathway is required for assembly and secretion of a superfamily of virulence-associated surface structures by a broad range of pathogens. The prototypical organelles assembled by this pathway are the P and type 1 pili expressed by uropathogenic Escherichia coli, the primary causative agent of urinary tract infections. P and type 1 pili are critical virulence factors, allowing binding and colonization of the kidney and bladder, respectively. The goals of this proposal are to probe the structure and function of the usher to gain an understanding of the molecular mechanisms governing pilus biogenesis across the outer membrane and to use the chaperone/usher pathway as a model system for understanding virulence factor secretion in Gram-negative bacteria. This proposal will test the hypothesis that the usher functions as a dimeric complex in the outer membrane to recognize, recruit and position chaperone-subunit complexes to catalyze the exchange of chaperone-subunit for subunit-subunit interactions, promote ordered pilus assembly, and allow secretion of the pilus fiber to the cell surface. The first specific aim will investigate the function of the usher as a catalytic machine and gated secretion channel for pilus biogenesis at the bacterial outer membrane. The second specific will determine the structural basis for pilus assembly and secretion at the outer membrane usher. This application will provide insights into mechanisms of protein secretion, organelle biogenesis, and the assembly of bacterial virulence factors.